Cell type annotation on data with all transcripts

workflowr

• Summary
• Checks
• Past versions

Last updated: 2025-10-20

Checks: 5 2

Knit directory: PIPAC_spatial/

This reproducible R Markdown analysis was created with workflowr (version 1.7.1). The Checks tab describes the reproducibility checks that were applied when the results were created. The Past versions tab lists the development history.

---

R Markdown file: uncommitted changes

The R Markdown file has unstaged changes. To know which version of the R Markdown file created these results, you’ll want to first commit it to the Git repo. If you’re still working on the analysis, you can ignore this warning. When you’re finished, you can run wflow_publish to commit the R Markdown file and build the HTML.

Environment: empty

Great job! The global environment was empty. Objects defined in the global environment can affect the analysis in your R Markdown file in unknown ways. For reproduciblity it’s best to always run the code in an empty environment.

Seed: set.seed(20240917)

The command set.seed(20240917) was run prior to running the code in the R Markdown file. Setting a seed ensures that any results that rely on randomness, e.g. subsampling or permutations, are reproducible.

Session information: recorded

Great job! Recording the operating system, R version, and package versions is critical for reproducibility.

Cache: none

Nice! There were no cached chunks for this analysis, so you can be confident that you successfully produced the results during this run.

File paths: absolute

Using absolute paths to the files within your workflowr project makes it difficult for you and others to run your code on a different machine. Change the absolute path(s) below to the suggested relative path(s) to make your code more reproducible.

absolute	relative
/home/hnatri/PIPAC_spatial/	.
/home/hnatri/PIPAC_spatial/code/PIPAC_colors_themes.R	code/PIPAC_colors_themes.R
/home/hnatri/PIPAC_spatial/code/plot_functions.R	code/plot_functions.R
/home/hnatri/PIPAC_spatial/cell_main_cluster_marker_annotations.tsv	cell_main_cluster_marker_annotations.tsv
/home/hnatri/PIPAC_spatial/cell_annotation_top_markers.tsv	cell_annotation_top_markers.tsv

Repository version: 2221fdb

Great! You are using Git for version control. Tracking code development and connecting the code version to the results is critical for reproducibility.

The results in this page were generated with repository version 2221fdb. See the Past versions tab to see a history of the changes made to the R Markdown and HTML files.

Note that you need to be careful to ensure that all relevant files for the analysis have been committed to Git prior to generating the results (you can use wflow_publish or wflow_git_commit). workflowr only checks the R Markdown file, but you know if there are other scripts or data files that it depends on. Below is the status of the Git repository when the results were generated:

Ignored files:
    Ignored:    .RData
    Ignored:    analysis/figure/
    Ignored:    cell_annotation_top_markers.tsv
    Ignored:    cell_annotation_top_markers_denoist.tsv
    Ignored:    cell_main_cluster_marker_annotations.tsv
    Ignored:    cell_main_cluster_marker_annotations_denoist.tsv
    Ignored:    cell_nonimmune_cluster_marker_annotations.tsv
    Ignored:    celltype_markers.tsv
    Ignored:    code/.Rhistory
    Ignored:    code/Proximity_analysis/.DS_Store
    Ignored:    code/Proximity_analysis/data/
    Ignored:    code/Proximity_analysis/output/
    Ignored:    code/RSC_latest_EDM_2025-08-06/
    Ignored:    code/pairwise_proximity.Rout
    Ignored:    immune_cluster_marker_annotations.tsv
    Ignored:    immune_cluster_marker_annotations_2ndpass.tsv
    Ignored:    main_cluster_marker_annotations.tsv
    Ignored:    nonimmune_cluster_marker_annotations.tsv
    Ignored:    nonimmune_cluster_marker_annotations_2ndpass.tsv

Untracked files:
    Untracked:  analysis/immune_annotation_cell.Rmd
    Untracked:  analysis/nonimmune_annotation_cell.Rmd
    Untracked:  code/denoist_chunk.R
    Untracked:  code/slurm.26422915.err
    Untracked:  code/slurm.26422915.out
    Untracked:  code/slurm.26435273.err
    Untracked:  code/slurm.26435273.out
    Untracked:  code/slurm.26571823.err
    Untracked:  code/slurm.26571823.out

Unstaged changes:
    Modified:   Rplots.pdf
    Modified:   analysis/Xenium_preprocess_wholecell.Rmd
    Modified:   analysis/add_metadata.Rmd
    Modified:   analysis/annotation_cell.Rmd
    Modified:   analysis/compare_cellular_nuclear.Rmd
    Modified:   analysis/compare_cellular_nuclear_denoist_annot.Rmd
    Modified:   analysis/index.Rmd
    Modified:   analysis/pca_variance_decomp.Rmd
    Modified:   analysis/splitting_samples.Rmd
    Modified:   code/PIPAC_colors_themes.R
    Modified:   code/anndata_to_seurat.R
    Modified:   code/denoist.R
    Modified:   code/parse_denoist_res.R
    Modified:   code/plot_functions.R
    Modified:   code/run_rscript.sh
    Modified:   code/seurat_to_anndata.R
    Modified:   code/update_metadata.R

Note that any generated files, e.g. HTML, png, CSS, etc., are not included in this status report because it is ok for generated content to have uncommitted changes.

---

These are the previous versions of the repository in which changes were made to the R Markdown (analysis/annotation_cell.Rmd) and HTML (docs/annotation_cell.html) files. If you’ve configured a remote Git repository (see ?wflow_git_remote), click on the hyperlinks in the table below to view the files as they were in that past version.

File	Version	Author	Date	Message
Rmd	7c46eb3	heinin	2025-09-29	Updated to denoised transcript data
html	7c46eb3	heinin	2025-09-29	Updated to denoised transcript data

---

Import packages

suppressPackageStartupMessages({
  library(workflowr)
  library(arrow)
  library(Seurat)
  library(SeuratObject)
  library(SeuratDisk)
  library(tidyverse)
  library(tibble)
  library(ggplot2)
  library(ggpubr)
  library(ggrepel)
  library(googlesheets4)
  library(workflowr)})

Environment variables and helper functions

setwd("/home/hnatri/PIPAC_spatial/")
set.seed(9999)
options(scipen = 99999)
options(ggrepel.max.overlaps = Inf)

source("/home/hnatri/PIPAC_spatial/code/PIPAC_colors_themes.R")
source("/home/hnatri/PIPAC_spatial/code/plot_functions.R")

Import data

# Copied to isilon /tgen_labs/banovich/PIPAC/Seurat
# /tgen_labs/banovich/PIPAC/Seurat/cell_merged_spatial_filtered_splitsamples_clustered_NC50_NN20_PC20_Seurat.rds.rds
seurat_data <- readRDS("/tgen_labs/banovich/PIPAC/Seurat/cell_merged_spatial_filtered_splitsamples_clustered_NN30_PC50_Seurat_denoIST.rds")
head(seurat_data@meta.data)

                                  orig.ident nCount_RNA nFeature_RNA
S21-24369_2A_TMA1_aaaafhkm-1_1 SeuratProject         34           22
S21-7951_5A_TMA1_aaabeech-1_1  SeuratProject        153           72
S21-7951_5A_TMA1_aaabidlm-1_1  SeuratProject         68           37
S21-7951_5A_TMA1_aaacanmk-1_1  SeuratProject         45           31
S21-7951_5A_TMA1_aaacnggd-1_1  SeuratProject        194           88
S21-7951_5A_TMA1_aaacoafb-1_1  SeuratProject         75           40
                                                      cell_id x_centroid
S21-24369_2A_TMA1_aaaafhkm-1_1 S21-24369_2A_TMA1_aaaafhkm-1_1   3120.635
S21-7951_5A_TMA1_aaabeech-1_1   S21-7951_5A_TMA1_aaabeech-1_1   3260.058
S21-7951_5A_TMA1_aaabidlm-1_1   S21-7951_5A_TMA1_aaabidlm-1_1   3167.581
S21-7951_5A_TMA1_aaacanmk-1_1   S21-7951_5A_TMA1_aaacanmk-1_1   3158.915
S21-7951_5A_TMA1_aaacnggd-1_1   S21-7951_5A_TMA1_aaacnggd-1_1   3176.202
S21-7951_5A_TMA1_aaacoafb-1_1   S21-7951_5A_TMA1_aaacoafb-1_1   3074.708
                               y_centroid transcript_counts
S21-24369_2A_TMA1_aaaafhkm-1_1   3213.421                34
S21-7951_5A_TMA1_aaabeech-1_1    5974.841               153
S21-7951_5A_TMA1_aaabidlm-1_1    6008.590                68
S21-7951_5A_TMA1_aaacanmk-1_1    6016.890                45
S21-7951_5A_TMA1_aaacnggd-1_1    6007.106               194
S21-7951_5A_TMA1_aaacoafb-1_1    5485.156                75
                               control_probe_counts genomic_control_counts
S21-24369_2A_TMA1_aaaafhkm-1_1                    0                      0
S21-7951_5A_TMA1_aaabeech-1_1                     0                      0
S21-7951_5A_TMA1_aaabidlm-1_1                     0                      0
S21-7951_5A_TMA1_aaacanmk-1_1                     0                      0
S21-7951_5A_TMA1_aaacnggd-1_1                     0                      0
S21-7951_5A_TMA1_aaacoafb-1_1                     0                      0
                               control_codeword_counts
S21-24369_2A_TMA1_aaaafhkm-1_1                       0
S21-7951_5A_TMA1_aaabeech-1_1                        0
S21-7951_5A_TMA1_aaabidlm-1_1                        0
S21-7951_5A_TMA1_aaacanmk-1_1                        0
S21-7951_5A_TMA1_aaacnggd-1_1                        0
S21-7951_5A_TMA1_aaacoafb-1_1                        0
                               unassigned_codeword_counts
S21-24369_2A_TMA1_aaaafhkm-1_1                          0
S21-7951_5A_TMA1_aaabeech-1_1                           0
S21-7951_5A_TMA1_aaabidlm-1_1                           0
S21-7951_5A_TMA1_aaacanmk-1_1                           0
S21-7951_5A_TMA1_aaacnggd-1_1                           0
S21-7951_5A_TMA1_aaacoafb-1_1                           0
                               deprecated_codeword_counts total_counts
S21-24369_2A_TMA1_aaaafhkm-1_1                          0           34
S21-7951_5A_TMA1_aaabeech-1_1                           0          153
S21-7951_5A_TMA1_aaabidlm-1_1                           0           68
S21-7951_5A_TMA1_aaacanmk-1_1                           0           45
S21-7951_5A_TMA1_aaacnggd-1_1                           0          194
S21-7951_5A_TMA1_aaacoafb-1_1                           0           75
                               cell_area nucleus_area nucleus_count
S21-24369_2A_TMA1_aaaafhkm-1_1  60.64485     33.68656             1
S21-7951_5A_TMA1_aaabeech-1_1  146.26110     32.06094             2
S21-7951_5A_TMA1_aaabidlm-1_1   75.36578     32.06094             1
S21-7951_5A_TMA1_aaacanmk-1_1   49.67188     18.78500             1
S21-7951_5A_TMA1_aaacnggd-1_1  126.75360     24.97141             1
S21-7951_5A_TMA1_aaacoafb-1_1   29.89344     14.90156             1
                                                                segmentation_method
S21-24369_2A_TMA1_aaaafhkm-1_1 Segmented by boundary stain (ATP1A1+CD45+E-Cadherin)
S21-7951_5A_TMA1_aaabeech-1_1  Segmented by boundary stain (ATP1A1+CD45+E-Cadherin)
S21-7951_5A_TMA1_aaabidlm-1_1  Segmented by boundary stain (ATP1A1+CD45+E-Cadherin)
S21-7951_5A_TMA1_aaacanmk-1_1  Segmented by boundary stain (ATP1A1+CD45+E-Cadherin)
S21-7951_5A_TMA1_aaacnggd-1_1  Segmented by boundary stain (ATP1A1+CD45+E-Cadherin)
S21-7951_5A_TMA1_aaacoafb-1_1  Segmented by boundary stain (ATP1A1+CD45+E-Cadherin)
                               num.blank           TMA percent.blank
S21-24369_2A_TMA1_aaaafhkm-1_1         0 MR_PIPAC-TMA1             0
S21-7951_5A_TMA1_aaabeech-1_1          0 MR_PIPAC-TMA1             0
S21-7951_5A_TMA1_aaabidlm-1_1          0 MR_PIPAC-TMA1             0
S21-7951_5A_TMA1_aaacanmk-1_1          0 MR_PIPAC-TMA1             0
S21-7951_5A_TMA1_aaacnggd-1_1          0 MR_PIPAC-TMA1             0
S21-7951_5A_TMA1_aaacoafb-1_1          0 MR_PIPAC-TMA1             0
                               nCount_cell_RNA nFeature_cell_RNA       Sample
S21-24369_2A_TMA1_aaaafhkm-1_1              34                22 S21-24369_2A
S21-7951_5A_TMA1_aaabeech-1_1              153                72  S21-7951_5A
S21-7951_5A_TMA1_aaabidlm-1_1               68                37  S21-7951_5A
S21-7951_5A_TMA1_aaacanmk-1_1               45                31  S21-7951_5A
S21-7951_5A_TMA1_aaacnggd-1_1              194                88  S21-7951_5A
S21-7951_5A_TMA1_aaacoafb-1_1               75                40  S21-7951_5A
                               leiden_0.5 leiden_1.0 leiden_1.5 leiden_2.0
S21-24369_2A_TMA1_aaaafhkm-1_1          9         10         13         16
S21-7951_5A_TMA1_aaabeech-1_1           3          6          2          7
S21-7951_5A_TMA1_aaabidlm-1_1           3          6          2          7
S21-7951_5A_TMA1_aaacanmk-1_1           3          6          2          7
S21-7951_5A_TMA1_aaacnggd-1_1           3          6          2          7
S21-7951_5A_TMA1_aaacoafb-1_1           5         26          7         33
                               Annotation Institution  Patient_ID Timepoint
S21-24369_2A_TMA1_aaaafhkm-1_1  EpiTumor1         NWH S21-NWH-012        12
S21-7951_5A_TMA1_aaabeech-1_1        Meso         NWH S21-NWH-011         0
S21-7951_5A_TMA1_aaabidlm-1_1        Meso         NWH S21-NWH-011         0
S21-7951_5A_TMA1_aaacanmk-1_1        Meso         NWH S21-NWH-011         0
S21-7951_5A_TMA1_aaacnggd-1_1        Meso         NWH S21-NWH-011         0
S21-7951_5A_TMA1_aaacoafb-1_1          M8         NWH S21-NWH-011         0
                               Tissue Location_Quadrant RPN
S21-24369_2A_TMA1_aaaafhkm-1_1  Tumor       Right Lower 012
S21-7951_5A_TMA1_aaabeech-1_1  Normal           Unknown 011
S21-7951_5A_TMA1_aaabidlm-1_1  Normal           Unknown 011
S21-7951_5A_TMA1_aaacanmk-1_1  Normal           Unknown 011
S21-7951_5A_TMA1_aaacnggd-1_1  Normal           Unknown 011
S21-7951_5A_TMA1_aaacoafb-1_1  Normal           Unknown 011
                                                      STUDY_SITE GENDER
S21-24369_2A_TMA1_aaaafhkm-1_1 Northwell Health Cancer Institute   Male
S21-7951_5A_TMA1_aaabeech-1_1  Northwell Health Cancer Institute   Male
S21-7951_5A_TMA1_aaabidlm-1_1  Northwell Health Cancer Institute   Male
S21-7951_5A_TMA1_aaacanmk-1_1  Northwell Health Cancer Institute   Male
S21-7951_5A_TMA1_aaacnggd-1_1  Northwell Health Cancer Institute   Male
S21-7951_5A_TMA1_aaacoafb-1_1  Northwell Health Cancer Institute   Male
                                                ETHNICITY      race DISEASESITE
S21-24369_2A_TMA1_aaaafhkm-1_1 Non-Hispanic or Non-Latino Caucasian Appendiceal
S21-7951_5A_TMA1_aaabeech-1_1  Non-Hispanic or Non-Latino Caucasian  Colorectal
S21-7951_5A_TMA1_aaabidlm-1_1  Non-Hispanic or Non-Latino Caucasian  Colorectal
S21-7951_5A_TMA1_aaacanmk-1_1  Non-Hispanic or Non-Latino Caucasian  Colorectal
S21-7951_5A_TMA1_aaacnggd-1_1  Non-Hispanic or Non-Latino Caucasian  Colorectal
S21-7951_5A_TMA1_aaacoafb-1_1  Non-Hispanic or Non-Latino Caucasian  Colorectal
                               ECOG ACTUALWEIGHT txstartdate
S21-24369_2A_TMA1_aaaafhkm-1_1    1         62.5        <NA>
S21-7951_5A_TMA1_aaabeech-1_1     0           86        <NA>
S21-7951_5A_TMA1_aaabidlm-1_1     0           86        <NA>
S21-7951_5A_TMA1_aaacanmk-1_1     0           86        <NA>
S21-7951_5A_TMA1_aaacnggd-1_1     0           86        <NA>
S21-7951_5A_TMA1_aaacoafb-1_1     0           86        <NA>
                                                          DXHISTOLOGY
S21-24369_2A_TMA1_aaaafhkm-1_1 8140/6-ADENOCARCINOMA, METASTATIC, NOS
S21-7951_5A_TMA1_aaabeech-1_1  8140/6-ADENOCARCINOMA, METASTATIC, NOS
S21-7951_5A_TMA1_aaabidlm-1_1  8140/6-ADENOCARCINOMA, METASTATIC, NOS
S21-7951_5A_TMA1_aaacanmk-1_1  8140/6-ADENOCARCINOMA, METASTATIC, NOS
S21-7951_5A_TMA1_aaacnggd-1_1  8140/6-ADENOCARCINOMA, METASTATIC, NOS
S21-7951_5A_TMA1_aaacoafb-1_1  8140/6-ADENOCARCINOMA, METASTATIC, NOS
                                            DXSITE initialdxdate LINETHERAPY
S21-24369_2A_TMA1_aaaafhkm-1_1      C18.1-Appendix          <NA>           2
S21-7951_5A_TMA1_aaabeech-1_1  C18.7-Sigmoid colon          <NA>           4
S21-7951_5A_TMA1_aaabidlm-1_1  C18.7-Sigmoid colon          <NA>           4
S21-7951_5A_TMA1_aaacanmk-1_1  C18.7-Sigmoid colon          <NA>           4
S21-7951_5A_TMA1_aaacnggd-1_1  C18.7-Sigmoid colon          <NA>           4
S21-7951_5A_TMA1_aaacoafb-1_1  C18.7-Sigmoid colon          <NA>           4
                                           PRIORTHERAPYTYPE priorcytoreduction
S21-24369_2A_TMA1_aaaafhkm-1_1 Chemotherapy, Multiple Agent                  0
S21-7951_5A_TMA1_aaabeech-1_1    Chemotherapy, Single Agent                 NA
S21-7951_5A_TMA1_aaabidlm-1_1    Chemotherapy, Single Agent                 NA
S21-7951_5A_TMA1_aaacanmk-1_1    Chemotherapy, Single Agent                 NA
S21-7951_5A_TMA1_aaacnggd-1_1    Chemotherapy, Single Agent                 NA
S21-7951_5A_TMA1_aaacoafb-1_1    Chemotherapy, Single Agent                 NA
                               lastcontactdate             furthertxall
S21-24369_2A_TMA1_aaaafhkm-1_1            <NA> Compassionate Use: PIPAC
S21-7951_5A_TMA1_aaabeech-1_1             <NA>                     <NA>
S21-7951_5A_TMA1_aaabidlm-1_1             <NA>                     <NA>
S21-7951_5A_TMA1_aaacanmk-1_1             <NA>                     <NA>
S21-7951_5A_TMA1_aaacnggd-1_1             <NA>                     <NA>
S21-7951_5A_TMA1_aaacoafb-1_1             <NA>                     <NA>
                               progressiondt vitalstatus1 deathdate
S21-24369_2A_TMA1_aaaafhkm-1_1            NA        Alive      <NA>
S21-7951_5A_TMA1_aaabeech-1_1             NA        Alive      <NA>
S21-7951_5A_TMA1_aaabidlm-1_1             NA        Alive      <NA>
S21-7951_5A_TMA1_aaacanmk-1_1             NA        Alive      <NA>
S21-7951_5A_TMA1_aaacnggd-1_1             NA        Alive      <NA>
S21-7951_5A_TMA1_aaacoafb-1_1             NA        Alive      <NA>
                                                                                               OFFSTUDYREASON
S21-24369_2A_TMA1_aaaafhkm-1_1                                                                           <NA>
S21-7951_5A_TMA1_aaabeech-1_1  Patient noncompliance after Week 14 - officially withdrew consent on 06AUG2021
S21-7951_5A_TMA1_aaabidlm-1_1  Patient noncompliance after Week 14 - officially withdrew consent on 06AUG2021
S21-7951_5A_TMA1_aaacanmk-1_1  Patient noncompliance after Week 14 - officially withdrew consent on 06AUG2021
S21-7951_5A_TMA1_aaacnggd-1_1  Patient noncompliance after Week 14 - officially withdrew consent on 06AUG2021
S21-7951_5A_TMA1_aaacoafb-1_1  Patient noncompliance after Week 14 - officially withdrew consent on 06AUG2021
                               offstudydate                      OFFTXREASON
S21-24369_2A_TMA1_aaaafhkm-1_1         <NA> Treatment Completed Per Protocol
S21-7951_5A_TMA1_aaabeech-1_1          <NA> Treatment Completed Per Protocol
S21-7951_5A_TMA1_aaabidlm-1_1          <NA> Treatment Completed Per Protocol
S21-7951_5A_TMA1_aaacanmk-1_1          <NA> Treatment Completed Per Protocol
S21-7951_5A_TMA1_aaacnggd-1_1          <NA> Treatment Completed Per Protocol
S21-7951_5A_TMA1_aaacoafb-1_1          <NA> Treatment Completed Per Protocol
                               offtxdate asascore1 asascore2 asascore3
S21-24369_2A_TMA1_aaaafhkm-1_1      <NA>     ASA 3     ASA 3     ASA 3
S21-7951_5A_TMA1_aaabeech-1_1       <NA>     ASA 3     ASA 3     ASA 3
S21-7951_5A_TMA1_aaabidlm-1_1       <NA>     ASA 3     ASA 3     ASA 3
S21-7951_5A_TMA1_aaacanmk-1_1       <NA>     ASA 3     ASA 3     ASA 3
S21-7951_5A_TMA1_aaacnggd-1_1       <NA>     ASA 3     ASA 3     ASA 3
S21-7951_5A_TMA1_aaacoafb-1_1       <NA>     ASA 3     ASA 3     ASA 3
                               lesionsize1 lesionsize2 lesionsize3 No_of_PIPACs
S21-24369_2A_TMA1_aaaafhkm-1_1           2           2           1            3
S21-7951_5A_TMA1_aaabeech-1_1            0           2           0            3
S21-7951_5A_TMA1_aaabidlm-1_1            0           2           0            3
S21-7951_5A_TMA1_aaacanmk-1_1            0           2           0            3
S21-7951_5A_TMA1_aaacnggd-1_1            0           2           0            3
S21-7951_5A_TMA1_aaacoafb-1_1            0           2           0            3
                               pipacdate1 pipacdate2 pipacdate3
S21-24369_2A_TMA1_aaaafhkm-1_1        YES        YES        YES
S21-7951_5A_TMA1_aaabeech-1_1         YES        YES        YES
S21-7951_5A_TMA1_aaabidlm-1_1         YES        YES        YES
S21-7951_5A_TMA1_aaacanmk-1_1         YES        YES        YES
S21-7951_5A_TMA1_aaacnggd-1_1         YES        YES        YES
S21-7951_5A_TMA1_aaacoafb-1_1         YES        YES        YES
                                               ascites1
S21-24369_2A_TMA1_aaaafhkm-1_1   Large volume (>500 mL)
S21-7951_5A_TMA1_aaabeech-1_1  Small volume (<= 500 mL)
S21-7951_5A_TMA1_aaabidlm-1_1  Small volume (<= 500 mL)
S21-7951_5A_TMA1_aaacanmk-1_1  Small volume (<= 500 mL)
S21-7951_5A_TMA1_aaacnggd-1_1  Small volume (<= 500 mL)
S21-7951_5A_TMA1_aaacoafb-1_1  Small volume (<= 500 mL)
                                               ascites2
S21-24369_2A_TMA1_aaaafhkm-1_1 Small volume (<= 500 mL)
S21-7951_5A_TMA1_aaabeech-1_1  Small volume (<= 500 mL)
S21-7951_5A_TMA1_aaabidlm-1_1  Small volume (<= 500 mL)
S21-7951_5A_TMA1_aaacanmk-1_1  Small volume (<= 500 mL)
S21-7951_5A_TMA1_aaacnggd-1_1  Small volume (<= 500 mL)
S21-7951_5A_TMA1_aaacoafb-1_1  Small volume (<= 500 mL)
                                               ascites3 transfusion1
S21-24369_2A_TMA1_aaaafhkm-1_1 Small volume (<= 500 mL)         None
S21-7951_5A_TMA1_aaabeech-1_1  Small volume (<= 500 mL)         None
S21-7951_5A_TMA1_aaabidlm-1_1  Small volume (<= 500 mL)         None
S21-7951_5A_TMA1_aaacanmk-1_1  Small volume (<= 500 mL)         None
S21-7951_5A_TMA1_aaacnggd-1_1  Small volume (<= 500 mL)         None
S21-7951_5A_TMA1_aaacoafb-1_1  Small volume (<= 500 mL)         None
                               transfusion2 transfusion3 ebl1 ebl2 ebl3 pci1
S21-24369_2A_TMA1_aaaafhkm-1_1         None         None    5    5    5   29
S21-7951_5A_TMA1_aaabeech-1_1          None         None   20   20    5   14
S21-7951_5A_TMA1_aaabidlm-1_1          None         None   20   20    5   14
S21-7951_5A_TMA1_aaacanmk-1_1          None         None   20   20    5   14
S21-7951_5A_TMA1_aaacnggd-1_1          None         None   20   20    5   14
S21-7951_5A_TMA1_aaacoafb-1_1          None         None   20   20    5   14
                               pci2 pci3 numcycles_pipacsurg cyclenum     age
S21-24369_2A_TMA1_aaaafhkm-1_1   28   24                   3        3 61.6427
S21-7951_5A_TMA1_aaabeech-1_1    13   10                   3        3 32.5749
S21-7951_5A_TMA1_aaabidlm-1_1    13   10                   3        3 32.5749
S21-7951_5A_TMA1_aaacanmk-1_1    13   10                   3        3 32.5749
S21-7951_5A_TMA1_aaacnggd-1_1    13   10                   3        3 32.5749
S21-7951_5A_TMA1_aaacoafb-1_1    13   10                   3        3 32.5749
                               lastfollowdate oscensor osmonths pfscensor
S21-24369_2A_TMA1_aaaafhkm-1_1           <NA>        1  17.9055         1
S21-7951_5A_TMA1_aaabeech-1_1            <NA>        1   4.4353         1
S21-7951_5A_TMA1_aaabidlm-1_1            <NA>        1   4.4353         1
S21-7951_5A_TMA1_aaacanmk-1_1            <NA>        1   4.4353         1
S21-7951_5A_TMA1_aaacnggd-1_1            <NA>        1   4.4353         1
S21-7951_5A_TMA1_aaacoafb-1_1            <NA>        1   4.4353         1
                               pfsmonths  Arm SUBJECT_STATUS EXPIRED_DATE
S21-24369_2A_TMA1_aaaafhkm-1_1   17.9055 Arm2           <NA>         <NA>
S21-7951_5A_TMA1_aaabeech-1_1     4.4353 Arm2           <NA>         <NA>
S21-7951_5A_TMA1_aaabidlm-1_1     4.4353 Arm2           <NA>         <NA>
S21-7951_5A_TMA1_aaacanmk-1_1     4.4353 Arm2           <NA>         <NA>
S21-7951_5A_TMA1_aaacnggd-1_1     4.4353 Arm2           <NA>         <NA>
S21-7951_5A_TMA1_aaacoafb-1_1     4.4353 Arm2           <NA>         <NA>
                               race_oncore DOSELEVEL_STD HEIGHT COMMENTS500
S21-24369_2A_TMA1_aaaafhkm-1_1        <NA>          <NA>               <NA>
S21-7951_5A_TMA1_aaabeech-1_1         <NA>          <NA>               <NA>
S21-7951_5A_TMA1_aaabidlm-1_1         <NA>          <NA>               <NA>
S21-7951_5A_TMA1_aaacanmk-1_1         <NA>          <NA>               <NA>
S21-7951_5A_TMA1_aaacnggd-1_1         <NA>          <NA>               <NA>
S21-7951_5A_TMA1_aaacoafb-1_1         <NA>          <NA>               <NA>
                               PRIORHIPECYN furthertxdate No_of_PIPACS
S21-24369_2A_TMA1_aaaafhkm-1_1         <NA>          <NA>         <NA>
S21-7951_5A_TMA1_aaabeech-1_1          <NA>          <NA>         <NA>
S21-7951_5A_TMA1_aaabidlm-1_1          <NA>          <NA>         <NA>
S21-7951_5A_TMA1_aaacanmk-1_1          <NA>          <NA>         <NA>
S21-7951_5A_TMA1_aaacnggd-1_1          <NA>          <NA>         <NA>
S21-7951_5A_TMA1_aaacoafb-1_1          <NA>          <NA>         <NA>
                               priortherapytype1 agedx dxtosxmonths
S21-24369_2A_TMA1_aaaafhkm-1_1              <NA>                   
S21-7951_5A_TMA1_aaabeech-1_1               <NA>                   
S21-7951_5A_TMA1_aaabidlm-1_1               <NA>                   
S21-7951_5A_TMA1_aaacanmk-1_1               <NA>                   
S21-7951_5A_TMA1_aaacnggd-1_1               <NA>                   
S21-7951_5A_TMA1_aaacoafb-1_1               <NA>                   
                               sxtoendtxmonths sxtooffstudy cytoreduction
S21-24369_2A_TMA1_aaaafhkm-1_1                                       <NA>
S21-7951_5A_TMA1_aaabeech-1_1                                        <NA>
S21-7951_5A_TMA1_aaabidlm-1_1                                        <NA>
S21-7951_5A_TMA1_aaacanmk-1_1                                        <NA>
S21-7951_5A_TMA1_aaacnggd-1_1                                        <NA>
S21-7951_5A_TMA1_aaacoafb-1_1                                        <NA>
                               priorsxyn Site_abbr
S21-24369_2A_TMA1_aaaafhkm-1_1      <NA>   S21-NWH
S21-7951_5A_TMA1_aaabeech-1_1       <NA>   S21-NWH
S21-7951_5A_TMA1_aaabidlm-1_1       <NA>   S21-NWH
S21-7951_5A_TMA1_aaacanmk-1_1       <NA>   S21-NWH
S21-7951_5A_TMA1_aaacnggd-1_1       <NA>   S21-NWH
S21-7951_5A_TMA1_aaacoafb-1_1       <NA>   S21-NWH

unique(seurat_data$leiden_0.5)

 [1]  9  3  5 14 16 11  6 17  1  8  0 10 12  2 13 15  4  7

DefaultAssay(seurat_data) <- "denoist_RNA"

DimPlot(seurat_data,
        group.by = "leiden_0.5",
        cols = pipac_cluster_20_col,
        reduction = "umap",
        raster = T,
        label = T) +
  coord_fixed(ratio = 1) +
  theme_minimal() +
  NoLegend()

Past versions of unnamed-chunk-3-1.png

Version	Author	Date
7c46eb3	heinin	2025-09-29

Marker information

gs4_deauth()
metadata  <- gs4_get("https://docs.google.com/spreadsheets/d/1sXXwOreLxjMSUoPt79c6jmaQpluWkaxA5P5HfDsed3I/edit?usp=sharing")
markers <- read_sheet(metadata, sheet = "Markers")

Feature expression

plot_features <- c("PTPRC",
                   "CD3D", "CD3E", "CD4", "CD8A", # T cells
                   "STAT4", "STAT3", "TIGIT", "GZMB",
                   "SELL", "CD19", # B cells
                   "CD68", "CD44", "MARCO", "APOE", # Macrophages
                   "C1QB", "C1QBP",
                   "MUC5AC", "NOTCH3", "MS4A1", "PGA5", # Lineage markers
                   "FN1", "DCN", "LUM", # Fibroblasts
                   "EGR3", "TP53", "JUN", "KIT", # Tumor
                   "SOX9", "RNF43", "EPCAM")

DotPlot(seurat_data,
        group.by = "leiden_0.5",
        features = plot_features,
        cols = c("azure", "tomato3")) +
  RotatedAxis()

Past versions of unnamed-chunk-5-1.png

Version	Author	Date
7c46eb3	heinin	2025-09-29

DefaultAssay(seurat_data) <- "RNA"
FeaturePlot(seurat_data,
            slot = "data",
            features = plot_features,
            order = T,
            ncol = 5,
            reduction = "umap",
            raster = T,
            cols = c("gray89", "tomato3")) &
  coord_fixed(ratio = 1) &
  theme_bw() &
  NoLegend()

Past versions of unnamed-chunk-6-1.png

Version	Author	Date
7c46eb3	heinin	2025-09-29

DenoIST

plot_features <- c("PTPRC",
                   "CD3D", "CD3E", "CD4", "CD8A", # T cells
                   "STAT4", "STAT3", "TIGIT", "GZMB",
                   "SELL", "CD19", # B cells
                   "CD68", "CD44", "MARCO", "APOE", # Macrophages
                   "C1QB", "C1QBP",
                   "MUC5AC", "NOTCH3", "MS4A1", "PGA5", # Lineage markers
                   "FN1", "DCN", "LUM", # Fibroblasts
                   "EGR3", "TP53", "JUN", "KIT", # Tumor
                   "SOX9", "RNF43", "EPCAM")

DefaultAssay(seurat_data) <- "denoist_RNA"
DotPlot(seurat_data,
        group.by = "leiden_0.5",
        features = plot_features,
        cols = c("azure", "tomato3")) +
  RotatedAxis()

FeaturePlot(seurat_data,
            slot = "data",
            features = plot_features,
            order = T,
            ncol = 5,
            reduction = "umap",
            raster = T,
            cols = c("gray89", "tomato3")) &
  coord_fixed(ratio = 1) &
  theme_bw() &
  NoLegend()

Past versions of unnamed-chunk-8-1.png

Version	Author	Date
7c46eb3	heinin	2025-09-29

Top cluster markers

Idents(seurat_data) <- seurat_data$leiden_0.5
cluster_markers <- FindAllMarkers(seurat_data,
                                  return.thresh = 0.01,
                                  logfc.threshold = 0.5,
                                  min.pct = 0.20,
                                  verbose = T)

table(cluster_markers$cluster)

  9   3   5  14  16  11   6  17   1   8   0  10  12   2  13  15   4   7 
134  89 115  54  42  60  98  74  20  86  84  71  68  60  53  22  50  56 

hist(cluster_markers$avg_log2FC, main = "", xlab = "avg_log2FC", breaks = 100)

Past versions of unnamed-chunk-9-1.png

Version	Author	Date
7c46eb3	heinin	2025-09-29

hist(cluster_markers$p_val, main = "", xlab = "p_val", breaks = 100)

Past versions of unnamed-chunk-9-2.png

Version	Author	Date
7c46eb3	heinin	2025-09-29

hist(cluster_markers$p_val_adj, main = "", xlab = "p_val_adj", breaks = 100)

Past versions of unnamed-chunk-9-3.png

Version	Author	Date
7c46eb3	heinin	2025-09-29

top_cluster_markers <- cluster_markers %>%
  arrange(dplyr::desc(avg_log2FC)) %>%
  group_by(cluster) %>%
  dplyr::slice(1:10)

create_dotplot_heatmap(seurat_object = seurat_data,
                       plot_features = unique(top_cluster_markers$gene),
                       group_var = "leiden_0.5",
                       group_colors = pipac_cluster_20_col,
                       column_title = "",
                       row_km = 5,
                       col_km = 5,
                       row.order = NULL,
                       col.order = NULL)

Past versions of unnamed-chunk-10-1.png

Version	Author	Date
7c46eb3	heinin	2025-09-29

Saving top markers and annotations

output_cluster_markers <- cluster_markers %>%
  arrange(dplyr::desc(avg_log2FC)) %>%
  group_by(cluster) %>%
  dplyr::slice(1:30)

output_cluster_markers <- merge(top_cluster_markers, markers, by.x = "gene", by.y = "Gene")

write.table(output_cluster_markers, "/home/hnatri/PIPAC_spatial/cell_main_cluster_marker_annotations.tsv",
            quote = F, row.names = F, sep = "\t")

# Saving DenoIST top markers by original annotation
Idents(seurat_data) <- seurat_data$Annotation
cluster_markers <- FindAllMarkers(seurat_data,
                                  return.thresh = 0.01,
                                  logfc.threshold = 0.5,
                                  min.pct = 0.20,
                                  verbose = T)

output_cluster_markers <- cluster_markers %>%
  arrange(dplyr::desc(avg_log2FC)) %>%
  group_by(cluster) %>%
  dplyr::slice(1:30)

output_cluster_markers <- merge(top_cluster_markers, markers, by.x = "gene", by.y = "Gene")

write.table(output_cluster_markers, "/home/hnatri/PIPAC_spatial/cell_annotation_top_markers.tsv",
            quote = F, row.names = F, sep = "\t")

Subsetting immune and non-immune cells for subclustering

seurat_data$Lineage <- ifelse(seurat_data$leiden_0.5 %in% c(5, 6),
                              "Immune", "TumorStroma")

immune_subset <- subset(seurat_data, subset = Lineage == "Immune")
nonimmune_subset <- subset(seurat_data, subset = Lineage == "TumorStroma")

saveRDS(immune_subset, "/scratch/hnatri/PIPAC/cell_immune_subset.rds")
saveRDS(nonimmune_subset, "/scratch/hnatri/PIPAC/cell_nonimmune_subset.rds")

# To build on command line, run Rscript -e "rmarkdown::render('annotation_cell.Rmd')"
# Then "mv *.html /home/hnatri/PIPAC_spatial/docs/"

Session information

sessionInfo()

R version 4.3.0 (2023-04-21)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 22.04.3 LTS

Matrix products: default
BLAS:   /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3 
LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.20.so;  LAPACK version 3.10.0

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

time zone: Etc/UTC
tzcode source: system (glibc)

attached base packages:
[1] grid      stats     graphics  grDevices utils     datasets  methods  
[8] base     

other attached packages:
 [1] ComplexHeatmap_2.18.0 viridis_0.6.3         viridisLite_0.4.2    
 [4] circlize_0.4.15       plyr_1.8.8            RColorBrewer_1.1-3   
 [7] googlesheets4_1.1.0   ggrepel_0.9.3         ggpubr_0.6.0         
[10] lubridate_1.9.2       forcats_1.0.0         stringr_1.5.0        
[13] dplyr_1.1.2           purrr_1.0.1           readr_2.1.4          
[16] tidyr_1.3.0           tibble_3.2.1          ggplot2_3.4.2        
[19] tidyverse_2.0.0       SeuratDisk_0.0.0.9021 Seurat_5.0.1         
[22] SeuratObject_5.0.1    sp_1.6-1              arrow_21.0.0.1       
[25] workflowr_1.7.1      

loaded via a namespace (and not attached):
  [1] RcppAnnoy_0.0.20       splines_4.3.0          later_1.3.1           
  [4] cellranger_1.1.0       polyclip_1.10-4        fastDummies_1.7.3     
  [7] lifecycle_1.0.3        rstatix_0.7.2          doParallel_1.0.17     
 [10] rprojroot_2.0.3        globals_0.16.2         processx_3.8.1        
 [13] lattice_0.21-8         hdf5r_1.3.8            MASS_7.3-60           
 [16] backports_1.4.1        magrittr_2.0.3         limma_3.58.1          
 [19] plotly_4.10.2          sass_0.4.6             rmarkdown_2.22        
 [22] jquerylib_0.1.4        yaml_2.3.7             httpuv_1.6.11         
 [25] sctransform_0.4.1      spam_2.9-1             spatstat.sparse_3.0-1 
 [28] reticulate_1.29        cowplot_1.1.1          pbapply_1.7-0         
 [31] abind_1.4-5            Rtsne_0.16             presto_1.0.0          
 [34] BiocGenerics_0.48.1    git2r_0.32.0           S4Vectors_0.40.2      
 [37] IRanges_2.36.0         irlba_2.3.5.1          listenv_0.9.0         
 [40] spatstat.utils_3.0-3   goftest_1.2-3          RSpectra_0.16-1       
 [43] spatstat.random_3.1-5  fitdistrplus_1.1-11    parallelly_1.36.0     
 [46] leiden_0.4.3           codetools_0.2-19       tidyselect_1.2.0      
 [49] shape_1.4.6            farver_2.1.1           stats4_4.3.0          
 [52] matrixStats_1.0.0      spatstat.explore_3.2-1 googledrive_2.1.0     
 [55] jsonlite_1.8.5         GetoptLong_1.0.5       ellipsis_0.3.2        
 [58] progressr_0.13.0       iterators_1.0.14       ggridges_0.5.4        
 [61] survival_3.5-5         foreach_1.5.2          tools_4.3.0           
 [64] ica_1.0-3              Rcpp_1.0.10            glue_1.6.2            
 [67] gridExtra_2.3          xfun_0.39              withr_2.5.0           
 [70] fastmap_1.1.1          fansi_1.0.4            callr_3.7.3           
 [73] digest_0.6.31          timechange_0.2.0       R6_2.5.1              
 [76] mime_0.12              colorspace_2.1-0       Cairo_1.6-0           
 [79] scattermore_1.2        tensor_1.5             spatstat.data_3.0-1   
 [82] utf8_1.2.3             generics_0.1.3         data.table_1.14.8     
 [85] httr_1.4.6             htmlwidgets_1.6.2      whisker_0.4.1         
 [88] uwot_0.1.14            pkgconfig_2.0.3        gtable_0.3.3          
 [91] lmtest_0.9-40          htmltools_0.5.5        carData_3.0-5         
 [94] dotCall64_1.0-2        clue_0.3-64            scales_1.2.1          
 [97] png_0.1-8              knitr_1.43             rstudioapi_0.14       
[100] rjson_0.2.21           tzdb_0.4.0             reshape2_1.4.4        
[103] nlme_3.1-162           curl_5.0.0             cachem_1.0.8          
[106] zoo_1.8-12             GlobalOptions_0.1.2    KernSmooth_2.23-21    
[109] parallel_4.3.0         miniUI_0.1.1.1         pillar_1.9.0          
[112] vctrs_0.6.2            RANN_2.6.1             promises_1.2.0.1      
[115] car_3.1-2              xtable_1.8-4           cluster_2.1.4         
[118] evaluate_0.21          magick_2.7.4           cli_3.6.1             
[121] compiler_4.3.0         rlang_1.1.1            crayon_1.5.2          
[124] future.apply_1.11.0    ggsignif_0.6.4         labeling_0.4.2        
[127] ps_1.7.5               getPass_0.2-4          fs_1.6.2              
[130] stringi_1.7.12         deldir_1.0-9           assertthat_0.2.1      
[133] munsell_0.5.0          lazyeval_0.2.2         spatstat.geom_3.2-1   
[136] Matrix_1.6-5           RcppHNSW_0.5.0         hms_1.1.3             
[139] patchwork_1.1.2        bit64_4.0.5            future_1.32.0         
[142] statmod_1.5.0          shiny_1.7.4            highr_0.10            
[145] ROCR_1.0-11            gargle_1.4.0           igraph_1.4.3          
[148] broom_1.0.4            bslib_0.4.2            bit_4.0.5